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Background of MAD2L1 antibody

The spindle checkpoint ensures the fidelity of chromosome segregation in mitosis and meiosis. In response to defects in the mitotic apparatus, it blocks the activity of the anaphase-promoting complex, a large ubiquitin ligase required for chromosome segregation. Recent studies indicate that the spindle checkpoint monitors both the attachment of chromosomes to the mitotic spindle and the tension across the sister chromatid generated by microtubules. Upon checkpoint activation, checkpoint protein complexes containing BubR1(Mad3), Bub3, Mad2 and Cdc20 directly bind to the anaphase-promoting complex and inhibit its ligase activity. Therefore, the checkpoint proteins form a complex intracellular signalling network to inhibit the anaphase-promoting complex.

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Western Blot and Immunoprecipitation: MAD-2 Antibody [NB100-563] - Detection of human MAD2 on HeLa whole cell lysate using NB100-563. MAD2 was also immunoprecipitated using NB100-564. Immunoprecipitated MAD2 was blotted using NB100-564.

Figure. Western blot using Affinity Purified anti-MAD2L1 antibody shows detection of a predominant band at ~24 kDa corresponding to MAD2L1 (arrowhead) present in Jurkat (lane 1) and HeLa (lane 2) whole cell lysates using the 800 nm channel (green). The identity of the higher molecular weight bands is unknown, although they may represent complexes of MAD2L1 with related binding proteins. Specific band reactivity is blocked when the antibody is pre-incubated with immunizing peptide (lanes 4 and 5 respectively) which completely blocks antibody staining. ~ 35 µg of lysate was separated on a 4-20% Tris-glycine gel by SDS-PAGE and transferred onto nitrocellulose. After blocking the membrane was probed with the primary antibody diluted to 1:1200. Incubation was 2h at room temperature followed by washes and reaction with a 1:10,000 dilution of IRDyeTM 800 conjugated Gt-a-Rabbit IgG [H&L] MXHu for 45 min at room temperature. Molecular weight markers were used for size comparison using the 700 nm channel (lane 3). IRDye TM 800 fluorescence image was captured using the Odyssey® Infrared Imaging System developed by LI-COR. IRDye is a trademark of LI-COR, Inc. Other detection systems will yield similar results.

Western blot using GeneTex Mab anti-MAD2L1 antibody (GTX10691) shows detection of a band at ~24 kDa (arrowhead) corresponding to MAD2L1 present in a HeLa whole cell lysate (lane 1). Approximately 75 µg of lysate was separated by 4-20% TG SDS-PAGE. After blocking, the membrane was probed overnight at 4°C with the primary antibody diluted to 1:200. The membrane was washed and reacted with a 1:5,000 dilution of IRDye™800 conjugated Sh-a-Mouse IgG [H&L] for 45 min at room temperature (800 nm channel, green). Molecular weight estimation was made by comparison to prestained MW markers in lane M (700 nm channel, red). IRDye™800 fluorescence image was captured using the Odyssey® Infrared Imaging System developed by LI-COR. IRDye is a trademark of LI-COR, Inc.

Western blot using MAD2L1 polyclonal antibody ( Cat # PAB10076 ) shows detection of a predominant band at ~24 kDa corresponding to MAD2L1 ( arrowhead ) present in Jurkat ( lane 1 ) and HeLa ( lane 2 ) whole cell lysates using the 800 nmchannel ( green ).The identity of the higher molecular weight bands is unknown, although they may represent complexes of MAD2L1 with related binding proteins.Specific band reactivity is blocked when the antibody is pre-incubated with immunizingpeptide ( lanes 4 and 5 respectively ) which completely blocks antibody staining.Approximately 35 µg of lysate was separated on a 4-20% Tris-glycine gel by SDS-PAGE and transferred onto nitrocellulose.After blocking the membrane was probed with the primary antibody diluted to 1:1200.Incubation was 2h at room temperature followed by washes andreaction with a 1:10,000 dilution of IRDyeTM800 conjugated Gt-a-Rabbit IgG [H&L] MXHu for 45 min at room temperature.Molecular weight markers were used for size comparison using the 700 nm channel ( lane 3 ). IRDyeTM800 fluorescence image was captured using the Odyssey® Infrared Imaging System developed by LI-COR.IRDye is a trademark of LI-COR, Inc.

Western blot using Mab anti-MAD2L1 antibody shows detection of a band at ~24 kDa (arrowhead) corresponding to MAD2L1 present in a HeLa whole cell lysate (lane 1). Approximately 75 µg of lysate was separated by 4-20% TG SDSPAGE. After blocking, the membrane was probed overnight at 4°C with the primary antibody diluted to 1:200. The membrane was washed and reacted with a 1:5,000 dilution of IRDye(TM)800 conjugated Sh-a-Mouse IgG [H&L] for 45 min at room temperature (800 nm channel, green). Molecular weight estimation was made by comparison to prestained MW markers in lane M (700 nm channel, red). IRDye(TM)800 fluorescence image was captured using the Odyssey(R) Infrared Imaging System developed by LI-COR. IRDye is a trademark of LI-COR, Inc. Other detection systems will yield similar results.

Immunoprecipitation of MAD2 from Jurkat with AM20288AF-N (Lane 1) or Mouse IgG (Lane 2). After immunoprecipitation with the antibody, immunocomplex was resolved on SDS-PAGE and immunoblotted with AM20288AF-N MAD2 antibody.

HEK293T cells were transfected with the pCMV6-ENTRY control (Left lane) or pCMV6-ENTRY MAD2L1 (Right lane) cDNA for 48 hrs and lysed. Equivalent amounts of cell lysates (5 ug per lane) were separated by SDS-PAGE and immunoblotted with anti-MAD2L1.

HEK293T cells were transfected with the pCMV6-ENTRY control (Left lane) or pCMV6-ENTRY MAD2L1 (RC203273, Right lane) cDNA for 48 hrs and lysed. Equivalent amounts of cell lysates (5 ug per lane) were separated by SDS-PAGE and immunoblotted with anti-MAD2L1.